LOCUS       sequence-273774-        4604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Integration and excision of kanamycin cassette for the E. coli 
            genome via CreI, SceI, and lamba Red system. Contains SacB 
            selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4604)
  AUTHORS   Wei Y, Deng P, Mohsin A, Yang Y, Zhou H, Guo M, Fang H
  TITLE     An electroporation-free method based on Red recombineering for 
            markerless deletion and genomic replacement in the Escherichia coli 
            DH1 genome.
  JOURNAL   PLoS One. 2017 Oct 24;12(10):e0186891. doi: 
            10.1371/journal.pone.0186891. eCollection 2017.
  PUBMED    29065183
REFERENCE   2  (bases 1 to 4604)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2017-10-24"; volume: "12(10):e0186891. doi"; pages: " 
            10.1371/journal.pone.0186891. eCollection 2017"
COMMENT     Sequence Label: pBDK
FEATURES             Location/Qualifiers
     source          1..4604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     443..462
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     659..676
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1068..1158
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        1165..1183
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1185..1202
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1186..1202
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1507..1526)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1518..2333
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             1518..2333
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1591..1610)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     complement(2519..2535)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2519..2535)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2536..2554)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             join(2866..2952,2953..4287)
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     rep_origin      join(4601..4604,1..542)
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
ORIGIN
        1 gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc
       61 ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg gcttggagga
      121 gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga cttcaagact
      181 aactcctcta aatcaattac cagtggctgc tgccagtggt gcttttgcat gtctttccgg
      241 gttggactca agacgatagt taccggataa ggcgcagcgg tcggactgaa cggggggttc
      301 gtgcatacag tccagcttgg agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat
      361 gagacaaacg cggccataac agcggaatga caccggtaaa ccgaaaggca ggaacaggag
      421 agcgcacgag ggagccgcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
      481 gccaccactg atttgagcgt cagatttcgt gatgcttgtc aggggggcgg agcctatgga
      541 aaaacggctt tgccgcggcc ctctcacttc cctgttaagt atcttcctgg catcttccag
      601 gaaatctccg ccccgttcgt aagccatttc cgctcgccgc agtcgaacga ccgagcgtag
      661 cgagtcagtg agcgaggaag cggaatatat cctgtatcac atattctgct gacgcaccgg
      721 tgcagccttt tttctcctgc cacatgaagc acttcactga caccctcatc agtgccaaca
      781 tagtaagcca gtatacactc cgctagcgct gatgtccggc ggtgcttttg ccgttacgca
      841 ccaccccgtc agtagctgaa caggagggac agctgataga aacagaagcc actggagcac
      901 ctcaaaaaca ccatcataca ctaaatcagt aagttggcag catcacccga cgcactttgc
      961 gccgaataaa tacctgtgac ggaagatcac ttcgcagaat aaataaatcc tggtgtccct
     1021 gttgataccg ggaagccctg ggccaacttt tggcgaaaat gagacgttga tcggcacgta
     1081 agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta
     1141 tcgagatttt caggagctag atcttaatac gactcactat agggtgtaaa acgacggcca
     1201 gtcaaaacgt cgtgagacag tttggtaagc ttggtaccca attggagctc actagtgaat
     1261 tcgtagcaca cgaggtctct tagggataac agggtaatga gttggtagct cttgatccgg
     1321 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     1381 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     1441 cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct tacataaaca
     1501 gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct aggccgcgat
     1561 taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat aatgtcgggc
     1621 aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag ttgtttctga
     1681 aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga ctaaactggc
     1741 tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct gatgatgcat
     1801 ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa gaatatcctg
     1861 attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg cattcgattc
     1921 ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag gcgcaatcac
     1981 gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat ggctggcctg
     2041 ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat tcagtcgtca
     2101 ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta ataggttgta
     2161 ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc ctatggaact
     2221 gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat ggtattgata
     2281 atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc taagaattaa
     2341 ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     2401 acatttcccc gaaaagtgcc acctgtaggg ataacagggt aattctccct atagtgagtc
     2461 gtaggatccc catggctgca ggcatgcctc gagaccaaac tgtctcacga cgttttgggt
     2521 catagctgtt tcctgccgct gagcaataac tagcgtcgac tagttcttta ggcccgtagt
     2581 ctgcaaatcc ttttatgatt ttctatcaaa caaaagagga aaatagacca gttgcaatcc
     2641 aaacgagagt ctaatagaat gaggtcgaaa agtaaatcgc gcgggtttgt tactgataaa
     2701 gcaggcaaga cctaaaatgt gtaaagggca aagtgtatac tttggcgtca ccccttacat
     2761 attttaggtc tttttttatt gtgcgtaact aacttgccat cttcaaacag gagggctgga
     2821 agaagcagac cgctaacaca gtacataaaa aaggagacat gaacgatgaa catcaaaaag
     2881 tttgcaaaac aagcaacagt attaaccttt actaccgcac tgctggcagg aggcgcaact
     2941 caagcgtttg cgaaagaaac gaaccaaaag ccatataagg aaacatacgg catttcccat
     3001 attacacgcc atgatatgct gcaaatccct gaacagcaaa aaaatgaaaa atatcaagtt
     3061 cctgaatttg attcgtccac aattaaaaat atctcttctg caaaaggcct ggacgtttgg
     3121 gacagctggc cattacaaaa cgctgacggc actgtcgcaa actatcacgg ctaccacatc
     3181 gtctttgcat tagccggaga tcctaaaaat gcggatgaca catcgattta catgttctat
     3241 caaaaagtcg gcgaaacttc tattgacagc tggaaaaacg ctggccgcgt ctttaaagac
     3301 agcgacaaat tcgatgcaaa tgattctatc ctaaaagacc aaacacaaga atggtcaggt
     3361 tcagccacat ttacatctga cggaaaaatc cgtttattct acactgattt ctccggtaaa
     3421 cattacggca aacaaacact gacaactgca caagttaacg tatcagcatc agacagctct
     3481 ttgaacatca acggtgtaga ggattataaa tcaatctttg acggtgacgg aaaaacgtat
     3541 caaaatgtac agcagttcat cgatgaaggc aactacagct caggcgacaa ccatacgctg
     3601 agagatcctc actacgtaga agataaaggc cacaaatact tagtatttga agcaaacact
     3661 ggaactgaag atggctacca aggcgaagaa tctttattta acaaagcata ctatggcaaa
     3721 agcacatcat tcttccgtca agaaagtcaa aaacttctgc aaagcgataa aaaacgcacg
     3781 gctgagttag caaacggcgc tctcggtatg attgagctaa acgatgatta cacactgaaa
     3841 aaagtgatga aaccgctgat tgcatctaac acagtaacag atgaaattga acgcgcgaac
     3901 gtctttaaaa tgaacggcaa atggtacctg ttcactgact cccgcggatc aaaaatgacg
     3961 attgacggca ttacgtctaa cgatatttac atgcttggtt atgtttctaa ttctttaact
     4021 ggcccataca agccgctgaa caaaactggc cttgtgttaa aaatggatct tgatcctaac
     4081 gatgtaacct ttacttactc acacttcgct gtacctcaag cgaaaggaaa caatgtcgtg
     4141 attacaagct atatgacaaa cagaggattc tacgcagaca aacaatcaac gtttgcgcct
     4201 agcttcctgc tgaacatcaa aggcaagaaa acatctgttg tcaaagacag catccttgaa
     4261 caaggacaat taacagttaa caaataaaaa cgcaaaagaa aatgccgata tcctattggc
     4321 attttctttt atttcttatc aacataaagg tgaatcccat agaaagcggg cagtgagcgc
     4381 aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc gatgcccttg
     4441 agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactaa catgagaatt
     4501 acaacttata tcgtatgggg ctgacttcag gtgctacatt tgaagagata aattgcactg
     4561 aaatctagaa atattttatc tgattaataa gatgatcttc ttga
//
